Early detection of cancer is a key feature in treating cancer patients. There are various possibilities of detecting cancerous cells in a biological sample and thus to diagnose an existing cancer or at least to estimate the likelihood of future cancer development. These different approaches comprise physical examination of cancer tissue, morphological characterization of cancerous cells, immunohistochemical staining and characterization of cellular structures such as membranes and the nucleus, measuring the expression of tumour specific factors, etc.
One possibility of detecting cancerous cells is so called DNA cytometry, which measures the amount of nuclear DNA in order to detect deviations from normal DNA content. It is assumed that if a cell as a consequence of mutagenic events comprises less or more DNA than a known standard, which is known to be of a non-cancerous, i.e. “healthy” or “normal” type, this deviation in DNA content can be indicative of major chromosomal rearrangements and thus ongoing cancer development.
Quantitation of nuclear DNA by nucleic acids specific stains is thus increasingly coming into practice in both research and clinical applications in the context of cancer diagnosis.
The measurement of nuclear DNA content by either flow or image cytometry is based inter alia on the assumptions that (i) the amount of stain bound to DNA is proportional to the amount of DNA present and that (ii) the optical signal generated from the stain by emission, absorption or transmission is proportional to the amount of stain.
One DNA specific staining procedure which is typically used for the purpose of measuring nuclear DNA by DNA image cytometry is an acid-based reaction named after Feulgen and Rossenbeck, often simply referred to as the Feulgen reaction. Actually the Feulgen reaction is a chromogenic reaction in which DNA is hydrolyzed by an acid to create a purine-free DNA (i.e. so-called apurinic acid, APA) with free aldehyde groups. These are then reacted with a Schiff's reagent containing a dye that binds covalently to the free aldehyde groups.
While the Feulgen reaction is specific for DNA, it suffers from various drawbacks. It is a time-consuming and rather elaborate reaction that needs to be carefully controlled and validated in order to yield reproducible and meaningful results. One problem arises from the fact that HCl, which is commonly used for the removal of the purine bases, hydrolyzes the APA into smaller fragments. However, fragmentation of the APA molecules leads to removal of these fragments from the cell nucleus and thus to loss of stainable DNA material.
Therefore, attempts have been made in the art to rely on a combination of morphological characterization, immunohistochemical stains and DNA stains for reliably detecting cancerous cells and diagnosing cancer.
US 2004/0197839 A1 discloses the concomitant use of two different type of stains for identifying cancerous cells in which one stain e.g. detects morphological characteristics whereas a second (immunological) stain e.g. measures the expression of a tumour-specific marker.
However, there is a continuing need for methods that allow a reliable, rapid and efficient way of measuring the amount of nuclear DNA and detection of cancerous cells and cancers.